Thrombocytes, or platelets, are circulating blood elements derived from the cytoplasm of a giant cell, the megakaryocyte. They are not strictly cells and do not possess a nucleus. They have important roles in haemostasis, clot retraction, repair of damaged blood vessels and inflammation, and they are generally present in a concentration of about 250×109 per liter blood. The survival time in blood is usually 8-10 days. They are small and disc-shaped with a diameter of about 2 μm and a volume of about 7 fl. A thrombocyte contains substances involved in blood clotting, which substances are stored in certain granules within the thrombocyte and are released upon activation of the thrombocyte, such as at a rift in a blood vessel.
Determining a thrombocyte count is often important in connection with treating a patient. This analysis may be needed for diagnosing a number of disorders. For example, a low thrombocyte count (less than 50×109 per liter blood) is indicative of thrombocytopenia which is a common reason for a predisposition for bleeding. Low thrombocyte concentrations may be due to reduced production, caused by e.g. damaged bone marrow as a result of aplastic anaemia, acute leukaemia, myeloma or cytostatica, reduced survival time of the thrombocytes or a change in the thrombocyte distribution, such as thrombocyte aggregation. On the other hand, an increased thrombocyte concentration may be a result of chronic inflammatory disorders (thrombocytose), such as rheumatic arthritis, or of independent production of thrombocytes in the bone marrow (thrombocytemia) and may lead to blood cloths, thrombosis.
It is desirable to enable analysis results to be obtained as quickly as possible in order to minimize waiting times for patients and enabling a physician to make a decision of treatment and diagnosis directly when making a first examination of the patient. It would therefore be preferable to provide an analysis method which may be quickly performed by the physician or a nurse without the need of sending a test away to a laboratory.
Today, a thrombocyte count is normally obtained with an automated Coulter counter, whereby the blood components (cells and thrombocytes) are identified by means of electrical conductance, or impedance. U.S. Pat. No. 4,240,029 discloses an apparatus for counting platelets and red blood cells by means of an aperture type transducer which is able to size discriminate between the platelets and the red blood cells.
Another automated method for counting platelets uses laser light scatter in a flow cytometer. The platelets are identified by their relatively small size as indicated by the measured light scatter. For instance U.S. Pat. No. 5,817,519 discloses an application of this method.
Other current ways of assessing platelet count are by utilising platelet specific anti bodies, such as disclosed in WO 00/25140, or by measuring the amount of released platelet granule protein, e.g. thrombospondin or β-thromboglobulin, in a sample, such as disclosed in U.S. Pat. No. 6,027,904.
Thrombocytes are also sometimes counted in a microscope in accordance with the Brecher-Cronkite method, whereby a blood sample is mixed with an ammonium oxalate solution after which the thrombocytes, which are visible as light dots with a dark rim, are counted in a phase-contrast microscope. Another known way of counting thrombocytes is by using the commercially available reagent Plaxan™ in combination with a counting chamber, such as a Bürker chamber. A counting chamber is provided with a grid dividing the chamber in well-defined small volumes. The thrombocyte count can then be determined by counting the number of thrombocytes per box in the grid. The thrombocyte count is obtained manually by an analyst, who needs to be experienced in performing the analysis in order to be able to perform a reliable analysis. These analysis are time-consuming. Further, since they are performed manually, the results of the analysis may vary depending on the person performing the analysis.
There is still a need to speed up and simplify existing automated methods for determining a thrombocyte count such that analysis may be provided at point of care.